Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immunodeficiency virus (HIV-1) isolates from 8 Ethiopian and 8 Swedish AIDS patients, none of them treated with antiviral drugs, were compared for sensitivity to azido-deoxy-thymidine (AZT), dideoxy-inosine (ddI) and interferon-alpha. HIV was isolated from peripheral blood mononuclear class, identified by Western blot and nucleotide sequencing, and passaged 1-3 times. Sensitivity to the 3 drugs, expressed as ED50s relative to positive controls, was determined by culturing HIV in the presence of drugs in a range of concentrations and assaying the supernatant for p24 antigen and the virus pellet for reverse transcriptase (RT). Dose-dependent anti-HIV activity for AZT was seen in the 8 Ethiopian isolates, and ED50s for p24 antigen and RT activity were correlated. 1 Ethiopian HIV isolate was sensitive to ddI, and another, to interferon-alpha. 1 Swedish HIV was resistant to AZT, and on analysis had a mutation from threonine to tyrosine at position 215. There were no significant differences between ED50s for interferon in the Swedish and Ethiopian HIVs. Combined data for each drug showed correlation between the p24 antigen and RT activities of the Ethiopian and Swedish HIVs. Since there was no resistance observed in the Ethiopian HIV to AZT or ddI, low-dose treatment would probably slow progression of HIV infection in Ethiopians, if these drugs could be made available for clinical trials.
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PMID:Response of Ethiopian human immunodeficiency virus type 1 isolates to antiviral compounds. 128 93

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient.
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PMID:Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine. 131 43

CPT-11, a recently developed topoisomerase I (Topo I) inhibitor, attracts the attention not only of basic researchers but also of clinicians because of its high antitumor activity. The CPT-11 resistant human lung cancer cell line, PC-7/CPT, showed 10-fold resistance compared to parental cell line, PC-7. The total activity of Topo I in the resistant cell line was one fourth that of the parental sensitive cell line. The Topo I from the resistant cells was also 5-fold more resistant to the inhibitory effect of CPT-11 than that of the parental cells. We speculated that the alteration of the Topo I gene may be responsible for the change in topoisomerase activity of the CPT-11 resistant cell line. Therefore, we analyzed the mutation of Topo I gene using the method of single strand conformation polymorphism of polymerase chain reaction and the reverse transcriptase. We divided Topo I cDNA into ten fragments which overlapped each other and covered whole coding sequences of the Topo I cDNA. We observed mobility shift of two fragments in the PC-7/CPT, suggesting the presence of some mutations in these fragments. We performed the direct-sequencing of these portions by the dideoxy chain termination method and observed an altered sequence having a G to A base change in PC-7/CPT. This base substitution results in replacement of the conserved threonine at 729 position with alanine. These results suggest that the point mutation of Topo I gene is related to the decreases of Topo I activity and the sensitivity to Topo I inhibitor in PC-7/CPT cells.
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PMID:Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line. 133 3

We have constructed an expression plasmid containing the portion of the Moloney murine leukemia virus genome encoding the reverse transcriptase (RT). When introduced into Escherichia coli this plasmid induces the synthesis of a 70-kDa protein. The RT made in E. coli differs from the viral protein only in that there are two new amino acids, methionine and glycine, substituted for the threonine found at the N terminus of the viral enzyme. Approximately half of the E. coli synthesized RT enzyme is soluble in cell extracts. This protein is active in an RT assay, and like the enzyme purified from virions, is more active in the presence of Mn2+ than Mg2+. We have also constructed a plasmid that induces the synthesis of an RT-integration protein fusion.
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PMID:Expression in Escherichia coli of a Moloney murine leukemia virus reverse transcriptase whose structure closely resembles the viral enzyme. 245 89

Mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) at codon 215 has been shown to play a significant role in resistance to zidovudine (AZT). Substitution of threonine with tyrosine or phenylalanine alone confers decreased susceptibility to the inhibitor. In this study we constructed a panel of 10 viruses with different amino acids at this codon, including 7 novel mutants, and assessed their susceptibilities to AZT. The majority of the new mutants were AZT sensitive, whereas the Thr-215-->Trp mutant was partially resistant (threefold less susceptible). A combination of the Thr-215-->Trp with the other AZT resistance mutations Lys-70-->Arg and Met-41-->Leu gave additive resistance. The Thr-215-->Phe virus was less AZT resistant than the Thr-215-->Tyr mutant, both on its own and when each was combined with the Met-41-->Leu mutant. These observations confirm the general hypothesis that increased bulk of the amino acid side chains at this position confers decreased AZT sensitivity. A leucine-to-valine substitution at codon 74 has previously been found to confer dideoxynucleoside resistance. We constructed mutants with five novel amino acid substitutions (Ala, Gly, Glu, Met, and Asp) at codon 74. Of these, only one (that with the Met substitution) retained enough RT activity to yield viable virus. It thus appears that there are severe structure-function constraints on the amino acid side chains at this position in the enzyme. The activities of the Leu-74-->Ala and Leu-74-->Met RT enzymes expressed in Escherichia coli appeared to have reduced susceptibility to ddGTP compared with the wild-type enzyme. The mutants described in this work may prove useful for correlation with structural studies of the human immunodeficiency virus type 1 RT.
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PMID:Mutagenic study of codons 74 and 215 of the human immunodeficiency virus type 1 reverse transcriptase, which are significant in nucleoside analog resistance. 751 65

We have selected a human immunodeficiency virus type 1 (HIV-1) mutant strain with a moderate (sevenfold) level of resistance to the nucleoside analog 2',3'-didehydro-2',3'-dideoxythymidine (D4T or stavudine). After serial passage of the HXB2 strain of HIV-1 in MT4 cells, a novel mutation involving two nucleotide substitutions in codon 75 of the viral reverse transcriptase, altering valine to threonine, was seen. When introduced into a wild-type HIV-1 background by site-directed mutagenesis, the T-75 mutation conferred cross-resistance to the dideoxynucleosides dideoxyinosine and dideoxycytosine as well as to 2',3'-didehydro-2',3'-dideoxycytosine.
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PMID:Novel mutation (V75T) in human immunodeficiency virus type 1 reverse transcriptase confers resistance to 2',3'-didehydro-2',3'-dideoxythymidine in cell culture. 752 29

The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.
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PMID:The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. 755 96

An abundant 95-kDa protein belonging to the low density lipoprotein receptor supergene family is essential for chicken oocyte growth by mediating the uptake of multiple plasma-borne yolk precursors. This receptor harbors at the amino terminus a cluster of eight tandemly arranged repeats typical of the ligand binding domains of members of this family and is designated low density lipoprotein receptor relative with 8 repeats (LR8). Here, we demonstrate by reverse transcriptase-polymerase chain reaction, Northern, and Western blot analyses that the chicken expresses two forms of LR8, which are generated by differential splicing of an exon encoding a serine- and threonine-rich region characteristic of LRs, termed O-linked sugar domain. The female germ cell of the chicken expresses extremely high levels of the short form of LR8 (LR8-), i.e. the 95-kDa protein; in contrast, somatic cells express lower but detectable levels of the form containing the O-linked sugar domain (LR8+). The main sites of LR8+ expression in the chicken are the heart and skeletal muscle, i.e. the same tissues were LR8 mRNAs predominate in mammals; in addition, in situ hybridization demonstrates that a significant amount of LR8+ is produced in the hen's ovarian follicular granulosa cells. We found no apparent functional difference between the two receptor forms; however, cell type-specific targeting of the multiple ligands of these receptors possibly relates to their respective expression on the cell surface.
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PMID:Chicken oocytes and somatic cells express different splice variants of a multifunctional receptor. 755 19

Human CD6 is a monomeric 105/130-kDa T cell surface glycoprotein that is involved in T cell activation. The apparent discrepancy between the size of the cytoplasmic domain in human (44 amino acids) and mouse (243 amino acids) CD6, led us to use reverse transcriptase-polymerase chain reaction of human peripheral blood lymphocyte mRNA to isolate cDNA clones that include the carboxyl-terminal coding region of human CD6. The nucleotide sequence of the longest human cDNA clone, CD6-PB1, predicts a protein of 668 amino acids with a 244-amino acid cytoplasmic domain similar in size to and possessing 71.5% amino acid sequence identity with the cytoplasmic domain of mouse CD6. This previously unrecognized 244-amino acid cytoplasmic domain does not have significant homology to any other known protein (except mouse CD6), but does possess two proline-rich motifs containing the SH3 domain-binding consensus sequence, a serine-threonine-rich motif repeated three times, three protein kinase C phosphorylation-site motifs, and 10 casein kinase-2 phosphorylation-site motifs. These sequences are likely to play a role in the ability of CD6-specific monoclonal antibodies to stimulate T cell proliferation. Full-length CD6 cDNA containing this cytoplasmic domain sequence encodes a monomeric 105/130-kDa protein that can be immunoprecipitated from the surface of transfected cells and comigrates upon SDS-PAGE with wild-type CD6 immunoprecipitated from PBL. We also isolated two alternatively spliced forms of human CD6 cDNA lacking sequences encoding membrane-proximal regions of the cytoplasmic domain which maintain the same reading frame as CD6-PB1. The short cytoplasmic domain of the previously reported human CD6-15 cDNA clone results from a deletion of a 20-bp segment through use of an alternative 3' splice site, resulting in a frame shift and premature termination of translation relative to the clones we have isolated. These data demonstrate that human CD6 possesses a large cytoplasmic domain containing sequence motifs that are likely to be involved in signal transduction upon stimulation of T cells through CD6 ligation.
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PMID:Human CD6 possesses a large, alternatively spliced cytoplasmic domain. 758 69

Transforming growth factor-beta 1 (TGF-beta 1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-beta receptors. We explored the possibility of inhibiting TGF-beta-mediated events in glomerular capillary endothelial cells using a TGF-beta type II receptor (T beta R-II) transdominant negative mutant. A mutant TGF-beta type II receptor (T beta R-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat T beta R-II cDNA as template. Since T beta R-II and TGF-beta type I receptor (T beta R-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type T beta R-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with T beta R-IIM, and four independent clones were expanded. That the T beta R-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface T beta R-IIM protein was shown by affinity cross-linking with 125I-TGF-beta 1. In wild-type endothelial cells, TGF-beta 1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 +/- 10% of control (n = 4). In transfected endothelial cells carrying T beta R-IIM, TGF-beta 1 stimulated [3H]thymidine incorporation to 131 +/- 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-beta 1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying T beta R-IIM. This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with T beta R-II signaling inhibits this process in glomerular capillary endothelial cells.
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PMID:Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor-beta receptors. 767 46


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